Mouse transplant models for evaluating the oncogenic risk of a self-inactivating XSCID lentiviral vector

Abstract

Hematopoietic stem cell gene therapy requires the use of integrating retroviral vectors in order to stably transmit a therapeutic gene to mature blood cells. Human clinical trials have shown that some vector integration events lead to disrupted regulation of proto-oncogenes resulting in disordered hematopoiesis including T-cell leukemia. Newer vectors have been designed to decrease the incidence of these adverse events but require appropriate pre-clinical assays to demonstrate safety. We have used two distinct mouse serial transplant assays to evaluate the safety of a self-inactivating lentiviral vector intended for use in X-linked severe combined immunodeficiency (XSCID) gene therapy trials. These experiments entailed 28 months of total follow-up and included 386 mice. There were no cases in which the XSCID lentiviral vector clearly caused hematopoietic malignancies, although a single case of B cell malignancy was observed that contained the lentiviral vector as a likely passenger event. In contrast, a SFFV-DsRed γ-retroviral vector resulted in clonal transformation events in multiple secondary recipients. Non-specific pathology not related to vector insertions was noted including T cell leukemias arising from irradiated recipient cells. Overall, this comprehensive study of mouse transplant safety assays demonstrate the relative safety of the XSCID lentiviral vector but also highlight the limitations of these assays.

Figure 1. Schematic representation of vectors and mouse transplant experiments.

(A) The self-inactivating lentiviral vector for XSCID and three gamma retroviral vectors that were used in this study are shown. The deleted U3 of the HIV-1 LTR was replaced with the 400 bp fragment from chicken beta-globlin insulator element (ΔU3+Ins). The codon optimized human IL2RG cDNA (hγcOPT), the eukaryotic elongation factor 1α promoter (EF1α); the central polypurine tract (cPPT), the Rev response element (RRE) and the psi packaging sequence (Ψ) are also shown. MFG-hγc is the γ- retroviral vector used in XSCID gene therapy trials. A MSCV vector expressing both a human γ_c cDNA and an IRES-GRP cassette is shown. The spleen focus-forming virus backbone expressing the DsRed fluorophore is also shown (SFFV-DsRed). Arrows indicate the transcription start sites for each vector. (B)A schematic design of the transplant experiments is shown. Bone marrow cells from γc^−/− or normal mice were transduced and were transplanted into lethally irradiated primary recipient mice of either of the indicated genotypes. Five to seven months later, transplanted mice were euthanized and bone marrow cells from each primary recipient were transplanted into two or three secondary recipient mice. The endpoints for these transplant groups are summarized.

Figure 2. Characterization of B cell leukemias in three secondary recipients that received bone marrow cells from one primary recipient in experiment 1 in the EF1α vector group.

(A) Flow cytometry analysis of spleen cells in an affected secondary recipient. Staining was done for surface expression of IgM (Y axis) and B220 (X axis) and percentages of cells in each gate are indicated. (B) Southern blot analysis of DNA from bone marrow and spleen cells using a vector probe and a single cutter enzyme for insertion site analysis. Bone marrow and spleen cell samples are indicated and identification of individual secondary mice is shown. The arrow indicated a clonal band derived from a single vector insertion in all three cases. (C) Inverse-PCR identification of the vector insertion event is shown. The vector is inserted in the first intron of the Pkn2 gene in the reverse genomic orientation. The location of the EcoRI sites used in the Southern analysis is shown as well as relative nucleotide distances. Exons are shown in boxes with corresponding numbers. (D) Quantitative real time PCR analysis of the Pkn2 transcripts in tumor cells and controls. Unfractionated total bone marrow cells (BL6/BM) was used as Pkn2 expression reference and the value was arbitrarily set as 1. Also shown are values for sorted IgM+ cells from spleen of a healthy C57BL6 mouse (BL6-IgM), sorted IgM+ leukemic cells from spleen of secondary recipient mouse (#203/IgM⁺), murine pre-B cell lymphoma cell line (70Z/3), and a murine mature-B cell lymphoma cell line (CH12). (E) Histology and immunohistochemistry for Pax5 expression are shown for a splenic B cell tumor from a secondary recipient are shown with size markers in the lower left of each panel.

Figure 3. Recipient origin of T-cell malignancies arising in the secondary recipients of the EF1a group in experiment 2.

Tumor cells derived from the spleen of transplanted mice were analyzed for CD4 and CD8 expression first (top panels). The abnormal CD4⁺ CD8⁺ leukemic cells were then gated and analyzed for CD45.1 (donor) and CD45.2 (recipient) marker expression. Virtually all CD4⁺CD8⁺ cells exclusively expressed CD45.2 and were therefore derived from the irradiated recipient mice.

Figure 4. Characterization of myeloid malignancies seen in the SFFV-treated secondary recipients.

(A) Hematologic characteristics of myeloid leukemias including peripheral leukocyte counts and spleen weights in individual mice at the time of euthanasia. (B)Flow cytometry analysis of splenic tumor cells from mouse #639. Staining for the Gr1 and Mac1 myeloid markers is shown. Gated normal cells (Gr1⁻ Mac1⁻ ) and tumor cells (Gr1⁺ Mac1⁺) were also analyzed for expression of the DsRed, vector-encoded marker. (C) Peripheral blood smear from a leukemic mouse with abnormal monocytic and granulocytic cells. (D) Southern blot analysis of DNA from bone marrow (B) and spleen (S) of six secondary mice that were derived from two primary recipients is shown. A clonal analysis for vector insertion sites was performed using a single cutter enzyme (BglII) and probed with DsRed cDNA probe. A common clonal pattern was noted from tumors derived from each primary recipient. All mice but #926 had clinical evidence of leukemia, however it was noted that #926 had already converted to a clonal pattern.

Figure 5. Skewing of myeloid differentiation in mice transplanted with SFFV-transduced cells.

(A) Percentage of peripheral blood cells in specific hematopoietic lineages at 30 weeks after transplantation. Each dot represents the percent of cells from a given lineage, as determined by flow cytometry, from a single primary transplant recipient from each of the vector groups listed on the X-axis. Statistical comparisons are shown for the indicated groups giving the p value. This analysis demonstrated a significant increase in cells expressing either the Gr1 or the Mac1 marker. (B) Percentage of DsRed+ peripheral blood cells in each lineage in the SFFV group of primary recipients at 30 weeks after transplant. Each line represents the profile from a single mouse and the lineages are indicated on the X-axis.